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Abstract

We report a simple and efficient colorimetric method to screen large numbers of bacterial
strains for UV- and X-radiation sensitivity. We used reference radiation-sensitive
and control strains of Escherichia coli K-12 to compare our colorimetric method to a standard clonogenic plating method.
Our colorimetric method was as accurate as the standard method and was superior in
terms of savings in supplies and man-hours.

Keywords:

Background

Studies on radiation-sensitive mutants of bacteria, e.g., Escherichia coli, have been invaluable in elucidating mechanisms of DNA repair (Augusto-Pinto et al.
f2003; Friedberg et al. 2006). However, it is common that one needs to screen, in time-consuming and expensive
fashion, large numbers of strains to find or quantitate a desired phenotype. With
this goal, we developed a resazurin-based assay using 96-well microtiter plates to
reduce the very significant time and expense normally associated with traditional
clonogenic (plating) assays for radiation sensitivity. We believe our new assay is
sensitive, rapid, robust and economical, and it should facilitate any studies where
the goal is to quickly and economically separate strains of differing radiation sensitivities,
e.g., mapping or transformation studies involving hundreds of strains, or studies
where large numbers of agents and concentrations would be tested for their impact
on radiation survival. Conversely, our assay can be used to test for factors or mutant
genotypes that might produce radiation resistance. The value of our new assay is directly
proportional to number of strains or conditions that need to be tested efficiently
and at low cost. Although we describe our assay using E. coli, this assay should be easily modifiable for use with other bacteria or higher organisms.

Resazurin is a purple, non-toxic, oxidation-reduction indicator that becomes pink
when reduced to resorufin by cellular oxidoreductases (Vega-Avila and Pugsley 2011). The concentration of viable cells in a suspension containing resazurin directly
determines the time-point for a visible conversion from purple to a pink color (Vega-Avila
and Pugsley 2011). Resazurin reduction tests have been used for decades to demonstrate bacterial and
yeast contamination of milk, and to determine chemical cytotoxicity and minimum inhibitory
concentration values for antibiotics (Bigalke 1984; Drummond and Waigh 2000; McNicholl et al. 2006; Sarker et al. 2007). Resazurin has been used in a few screening studies for radiation sensitivity of
mammalian cells (Gil et al. 2011; Seideman et al. 2010), however, to our knowledge, a resazurin-based assay for use in screening bacterial
strains for radiation sensitivity has not been described. As in other resazurin-based
studies, the readout in our assay is colorimetric and the rate of color change is
directly proportional to the number of viable cells in the initial suspension. Compared
to a DNA repair proficient, parental, control strain, the time-point for color conversion
is extended in suspensions of cells that are more sensitive to radiation and have
relatively fewer viable cells in the irradiated cell suspension. We developed this
technique so that one could visually scan hundreds of microtiter wells quickly. We
show that the visual results can be quantified with a microplate reader, but this
is not a requirement. Visual inspection will suffice to easily identify strains that
are more sensitive to radiation, i.e., their wells show more purple or less pink color
than the control strain after a set time of incubation.

We report on the reliability of our colorimetric assay by testing (i) the radiation
dosimetry among the 96 wells of a microtiter plate, (ii) the resazurin color change
for reference radiation-sensitive and -resistant strains of E. coli after both UV- and X-irradiation, and (iii) the sensitivity of our colorimetric assay
(an indirect measure of cell survival) in comparison with a clonogenic assay (a more
traditional and direct measure of cell survival) for differentiating a set of reference
E. coli strains based on their radiation sensitivities. We also report an estimate of the
cost savings in using the colorimetric assay vs. the clonogenic assay.

Results and discussion

First, we determined the well-to-well variation in X- and UV-radiation dosimetry in
our 96-well microtiter plates. We used chemical dosimetry to determine the mean X-radiation
dose rate over 288 wells (3 × 96), which was 17.96 ± 0.02 Gy min-1. The well-to-well variation of chemical dosimeter readings, which are directly proportional
to X-ray dose rates, is shown in Figure 1A. Although, we measured our UV radiation dose rate at 1.42 J m-2 s-1 for the entire irradiated field, we used our resazurin-based bioassay to assess the
mean effect of UV radiation on the cell suspensions in 576 wells (6 × 96), and this
was 0.77 ± 0.02 A492 units. The well-to-well variation in UV-radiation dose rate is represented by the
A492 values shown in Figure 1B.

Figure 1.X- and UV-radiation dose rates within the 96 wells of a microtiter plate. (A) The absorbance within each well was determined at 304 nm (A304) in triplicate experiments using a chemical dosimeter and plotted. The rows (letters)
and column (numbers) in the graph associate data with individual wells in the microtiter
plate. The average dose rate for 160 kV X-rays was determined from (ΔA304)(280 Gy min-1) to be 17.96 Gy/min (sd = 0.02), and plates received a dose of 54 Gy. (B) A492 values for resazurin absorbance (indicating the cellular metabolic activity within
each well) were determined from 6 experiments using a bioassay. Plates received a
UV radiation dose of 50 J m-2. Bioassay A492 values were averaged and plotted. The mean value over 96 wells was 0.77 (sd = 0.02)
A492 units.

Second, we visually assessed the color change after UV- or X-irradiation for 17 E. coli K-12 isogenic reference strains (Table 1), the DNA repair proficient, parental, control strain, SR749, and 16 others with
single, radiation-sensitizing mutations at the lexA, polA, radA, radC, recA, recB, recC, recF, recG, recJ, recN, ruvA, ruvB, umuC,
uvrD, or ruvC genes. During these experiments, we consistently were able to visually differentiate
the 16 “radiation-sensitive” strains compared to the parental control strain based
on culture color. An example of the color differential is shown in Figure 2.

The third test of the resazurin-based assay involved plotting the radiation sensitivity
data (direct measure) for our set of 17 reference E. coli strains against resazurin/resorufin absorbance values (indirect measure) in Figures 3A and 3B. Cell surviving fractions of UV- and X-irradiated cells were determined using a
clonogenic assay. Irradiated or non-irradiated cells were plated onto duplicate LB
agar plates. After overnight incubation at 37°C, colonies were counted and cell-surviving
fractions were calculated. Resazurin/resorufin absorbance values (A492) were attained from the colorimetric assay plates using a microplate reader. The
results of the colorimetric and clonogenic assays are shown in Table 2, and indicate a similar ability of each assay to differentiate radiation-sensitive
strains from the parental, control strain. Figures 3A and 3B confirm that irradiated strains showing lower surviving fractions (i.e., more sensitive
to radiation than the parental, control strain) also showed higher A492 values (i.e., their irradiated cell suspensions showed less metabolic activity than
the parental, control strain). We plotted the mean surviving fraction and A492 data (± 2 sem) for the parental, control strain (WT) to produce a gray-shaded box
in the upper left-hand corner of each graph. Mean ± sd data for reference test strains
that did not fall within the shaded box were considered significantly different from
the parental, control strain in their A492 values (Kruskal-Wallis one way ANOVA on Ranks: X-radiation H = 46.891 P < 0.001,
UV-radiation H = 47.370 P < 0.001). Under these constraints, only X-irradiated umuC cells were not different from the control strain, which was verified with a t-test (t = 1.912, P = 0.128). These results for umuC are consistent with published results for x-radiation sensitivity (Sargentini and
Smith 1986). In addition, we performed a t-test comparing the nearest “sensitive strain”, recJ in this case, to the parental control strain to confirm that it was statistically
different (t = 4.874, P = 0.008). For UV radiation, all test strains were significantly
different from the parental, control strain and the nearest “sensitive strains” were
verified with t-tests, umuC (t = 3.158, P = 0.025) and recJ (t = 4.775, P = 0.009). Although the data in Figures 3A and 3B suggest one could use our colorimetric assay to quantitatively differentiate E. coli strains on the basis of their radiation sensitivity, our focus was to develop a screening
assay that would allow simple and rapid differentiation of radiation-sensitive strains
from a parental, control strain.

Figure 3.Comparison of colorimetric and clonogenic assays for radiation-sensitive phenotype
of E. coli reference strains. Mutant strains listed in Table 1 were compared with the isogenic parental control strain (SR749, WT) for recovery
from radiation treatment. (A) X-radiation, 250 Gy. (B) UV-radiation, 100 J m-2. All data points are means from triplicate experiments. Standard deviations (horizontal
bars displaying variation in absorbance values and vertical bars displaying variation
in surviving fraction values) are shown. The gray-shaded boxes represent the mean ± 2
sem for the WT strain. Any strain data points that fall outside of the gray-shaded
boxes are significantly different (Kruskal-Wallis one way ANOVA on Ranks, P <0.05)
in their absorbance and surviving fraction values from the WT strain.

We compared our two assays for time and cost (Table 3), and found the colorimetric assay (compared to the clonogenic assay) would save
about $1200 and 7 days of work per 96 E. coli strains tested, without considering technician pay. Therefore, our colorimetric method
was superior in terms of man-hours, pipetting steps and expense for supplies when
compared to the clonogenic assay.

Conclusions

In summary, we have described a novel, resazurin-based colorimetric method for high-throughput
screening of E. coli strains for radiation sensitivity. This assay is easy to follow, depends on many
fewer pipetting steps, is highly economical in terms of man-hours and supplies, and
provides results that compare well with standard, more expensive and time-consuming
clonogenic assays.

UV radiation was supplied by an 8-W germicidal lamp (GE, G8T5) emitting primarily
at 254 nm. The UV radiation dose rate was 1.42 J m-2 s-1 at the base of a microtiter plate (47 cm below the lamp) using a germicidal photometer
(Model IL1700, International Light, Inc.). However, we used a bioassay to test for
uniformity of dose rate across the 96 wells of a microtiter plate. For this purpose,
E. coli strain SR749 was grown overnight in 5 ml Luria-Bertani (LB) broth (Miller 1972) supplemented with 1% glucose for 15–17 h (in a tube roller for aeration) to a stationary-phase
cell concentration of ~1 × 109 colony-forming units (CFU) per ml. Cultures were diluted ~15-fold to an optical density
at 600 nm (OD600) of 0.03 (NanoDrop 2000c; Fisher; corresponding to 6.4 × 107 CFU/ml) with 67 M NaK phosphate buffer (PB). A 50-μl cell volume was placed into
each well of six 96-well microtiter plates and these were UV-irradiated with a dose
of 50 J m-2. After irradiation, 150 μl of LB and 10 μl of 0.675% resazurin (Difco) solution were
added to each well and plates were incubated at 37°C for 4.5 h. To compare the UV
radiation doses received in each of the 96 wells of the microtiter plates, we determined
the development of pink color (related to cell viability) by measuring A492 values (LabSystems MultiSkan MCC/340).

To simultaneously measure radiation sensitivity of E. coli strains by resazurin and clonogenic assays, cells were prepared as above, but 300-μl
volumes of cell suspensions (~6.4 × 107 CFU/ml) were placed in wells in two separate 96-well microtiter plates. One plate
was X-irradiated (250 Gy), the other was UV irradiated (100 J m-2). After irradiation, cells were re-pipetted (3x) and 250 μl of cells from each well
were removed and saved for the clonogenic assay (along with a sample of non-irradiated
cells). The remaining 50-μl volume of cells was mixed with LB/resazurin solution and
incubated as above. The incubation time (4.5 h) was optimized in pilot experiments
to consistently be able to visually differentiate cultures of control radiation-resistant
strains (bright pink color) from cultures of radiation-sensitive strains (purple color).
Once incubation was complete, A492 values were determined as above. Cell surviving fractions of UV- and X-irradiated
cells were determined using our clonogenic assay by plating the cells saved from the
microtiter plates used in the colorimetric assay. Irradiated or non-irradiated cells
were spread onto duplicate LB agar plates, either directly or after dilution in PB.
After overnight incubation at 37°C, colonies were counted to determine the colony
forming units per ml (CFU/ml) values for the non-irradiated and irradiated cell suspensions.
The CFU/ml value for a cell suspension was determined by multiplying the mean number
of colonies per plate by the dilution factor. The cell surviving fraction was determined
as the ratio of the CFU/ml value after each radiation dose divided by the CFU/ml value
for non-irradiated (control) cells. Experiments were completed in triplicate to determine
the mean surviving fraction ± sd for each radiation dose.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

NJS and DAH designed the study, DAH performed experiments; DAH collected and analyzed
data; NJS provided reagents, supplies and technical support; DAH and NJS wrote and
approved the manuscript.